Multiplex microparticle system

ABSTRACT

Arrays of microparticle populations, each population labeled with a single fluorescent dye, are provided for use in multiplex assays. The populations form a virtual multidimensional array wherein each microparticle is identified by fluorescence intensity in two different fluorescence detection channels. The arrays are useful in a variety of assays, including multiplex, multi-analyte assays for the simultaneous detection of two or more analytes by, for example, flow cytometry, and a labeling reagents in, for example, microscopy. The use of singly-dyed microparticles to form multidimensional arrays greatly simplifies the creation of multiplex assays.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 11/404,348, filed Apr. 14, 2006, which issued as U.S. Pat. No.7,507,588 on Mar. 24, 2009, and which claims priority to U.S.provisional application No. 60/673,471, filed Apr. 20, 2005; thedisclosures of both of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to arrays of labeled microparticles. Sucharrays are particularly useful in multiplex assays, such as biologicaldetection assays and, more particularly, in the fields of flow cytometryand fluorescence microscopy.

2. Description of Related Art

Flow cytometers are well known analytical tools that enable thecharacterization of particles on the basis of light scatter and particlefluorescence. In a flow cytometer, particles are individually analyzedby exposing each particle to an excitation light, typically one or morelasers, and the light scattering and fluorescence properties of theparticles are measured. Particles, such as molecules, analyte-boundbeads, individual cells, or subcomponents thereof, typically are labeledwith one or more spectrally distinct fluorescent dyes, and detection iscarried out using a multiplicity of photodetectors, one for eachdistinct dye to be detected. Flow cytometers are commercially availablefrom, for example, BD Biosciences (San Jose, Calif.).

Early in the development of flow cytometry, it was recognized thatvarious types of ligand binding assays could be carried out usingmicroparticles (beads) coated with one member of a binding pair. Forexample, immunoassays can be carried out in a sandwich hybridizationassay format using beads coated with an analyte-specific binding agent,such as a monoclonal antibody (mAb), as a capture reagent, and a secondanalyte-specific binding agent, again typically a mAb, labeled with afluorophore as a reporter reagent. The coated beads and reporters areincubated with a sample containing (or suspected of containing) theanalyte of interest to allow for the formation of bead-analyte-reportercomplexes. Analysis by flow cytometry enables both detecting thepresence of bead-analyte-reporter complexes and simultaneously measuringthe amount of reporter fluorescence associated with the complex as aquantitative measure of the analyte present in the sample.

It was also recognized early in the development of flow cytometry thatthe simultaneous analysis of multiple analytes in a sample could becarried out using a set of distinguishable beads, each type of beadcoated with a unique analyte-specific binding agent. The bead set andfluorescently labeled reporter reagents, one for each species of analyteto be detected, are incubated with a sample containing the analytes ofinterest to Is allow for the formation of bead-analyte-reportercomplexes for each analyte present, and the resulting complexes areanalyzed by flow cytometry to identify and, optionally, quantify theanalytes present in the sample. Because the identity of the analytebound to the complex is indicated by the identity of the bead, multipleanalytes can be simultaneously detected using the same fluorophore forall reporter reagents. A number of methods of making and using sets ofdistinguishable microparticles have been described in the literature.

UK Patent No. 1 561 042, published Feb. 13, 1980, and Fulwyler andMcHugh, 1990, Methods in Cell Biology 33:613-629, describe the use ofmultiple microparticles distinguished by size, wherein each sizemicroparticle is coated with a different target-specific antibody.

Tripatzis, European Patent No. 0 126,450, published Nov. 28, 1984 (seealso corresponding Canadian Patent 1 248 873), describesmulti-dimensional arrays of microparticles formed by labelingmicroparticles with two or more fluorescent dyes at varyingconcentrations. Microparticles in the array are uniquely identified bythe levels of fluorescence dyes. Tripatzis describes the use of sucharrays for the simultaneous detection a large numbers of analytes in asample by flow cytometry, and, further, describes their use as labels inmicroscopy.

U.S. Pat. Nos. 4,499,052 and 4,717,655, Entitled: “Method and Apparatusfor Distinguishing Multiple Subpopulations of Cells”, issued Feb. 12,1985, and Jan. 5, 1988, respectively, describe the use of microparticlesdistinguishably labeled with two different dyes, wherein themicroparticles are identified by separately measuring the fluorescenceintensity of each of the dyes.

Both one-dimensional and two-dimensional arrays for the simultaneousanalysis of multiple analytes by flow cytometry are availablecommercially. Examples of one-dimensional arrays of singly dyed beadsdistinguishable by the level of fluorescence intensity include the BD™Cytometric Bead Array (CBA) (BD Biosciences, San Jose, Calif.) andCyto-Plex™ Flow Cytometry microspheres (Duke Scientific, Palo Alto,Calif.). An example of a two-dimensional array of beads distinguishableby a combination of fluorescence intensity (five levels) and size (twosizes) is the QuantumPlex™ microspheres (Bangs Laboratories, Fisher,Ind.). An example of a two-dimensional array of doubly-dyed beadsdistinguishable by the levels of fluorescence of each of the two dyes isdescribed in McDade and Fulton, April 1997, Medical Device & DiagnosticIndustry; and Fulton et al., 1997, Clinical Chemistry 43(9):1749-1756.

Each of the microparticle arrays described above has disadvantages thatlimit their utility. Arrays based on different size microparticles areproblematical because the amount of capture reagent that can be bound toa microparticle, which affects the sensitivity and dynamic range of theassay, is dependent on the particle size. Thus, to obtain uniform assayperformance for all analytes, it is desirable to use microparticles ofuniform size. One-dimensional arrays based on differences in thefluorescent intensity of a single dye typically are limited to about 10different microparticle populations. Although useful for a wide range ofassays, it is desirable to have more distinct microparticle populationsto enable the simultaneous detection of greater numbers of analytes.Two-dimensional arrays based on differences in the fluorescenceintensities of two distinct dyes enable much larger arrays, but aresignificantly more difficult to manufacture, and increase the difficultyin subsequent data analysis.

SUMMARY OF THE INVENTION

The present invention relates to multidimensional arrays formed frompopulations of singly dyed microparticles. The use of singly dyedmicroparticles to form multidimensional arrays greatly simplifies thecreation of multiplex assays, yet still provides most of the advantagesarrays formed from multiply dyed microparticles.

Multidimensional arrays of the present invention contain a plurality ofsets of microparticle populations, wherein different populations withina set are labeled, using the same fluorophore, at a plurality ofdiscrete fluorescence levels, and populations in different sets arelabeled with different fluorophores, wherein the emission of eachfluorophore, as measured in the same two detection channels, exhibitsdistinct relative amounts of emission in the two detection channels. Inpreferred embodiments, the array contains at least three sets ofmicroparticle populations, more preferably at least four, and morepreferably at least five.

The present invention is based on the surprising discovery that by usingspectrally similar fluorophores having overlapping emission spectra,practical multidimensional arrays can be created from a plurality ofone-dimensional arrays, each based on differences in the fluorescentintensity of a single fluorophore, and that such arrays created fromthree or more one-dimensional arrays can be analyzed using two detectionchannels. The overlapping emission spectra of the fluorophores enablethe detection of emission from each of the fluorophores using the sametwo detection channels.

The fluorescence properties of the multidimensional array of theinvention enable the identification of the microparticles in eachpopulation by exposing the array to excitation light and measuring thefluorescence of each microparticle in each of the two detectionchannels. The resulting fluorescence data can be plotted in atwo-dimensional dot-plot, plotting intensity of the two detectionchannels on the two axes, as is routinely used in flow cytometry. Eachpopulation will appear as cluster uniquely positioned in thetwo-dimensional dot plot.

The breadth of the emission spectrum of a typical fluorophore isnormally regarded as an undesirable property. For example, in flowcytometry, fluorescent dyes are selected where possible such that theemission spectra overlap minimally, and different detector channels areused to detect different dyes. To maximize detection sensitivity, eachdetector channel is selected such that, as much as possible, itcorresponds to the emission maximum of the single dye that it isintended to detect. Emission detected from the other dyes, caused by thebreadth of the other dye's emission spectrum, often referred to as“spillover” or “crosstalk”, is undesirable and interferes with theindependent measurement of dye fluorescence in these previouslydescribed methods.

In contrast, the present invention makes use of the breadth of theemission spectrum, and the resulting emission in two detection channels,to distinguish the fluorophores. The emission spectra of differentfluorophores having similar emission peaks will overlap each of the twochannels to differing degrees and, consequently, will exhibit differentrelative emission in the two channels. Thus, both microparticlepopulations labeled with different dyes (i.e., in different sets) andmicroparticle populations labeled with different amount of the same dye(i.e., in the same set) can distinguished by the different emissionintensities in the two detector channels.

The microparticle arrays of the present invention can be usedessentially in any application in which multiplex particle arrays areused or are useful, including applications in which the microparticlesare used a solid substrates for ligand binding assays or as labelingreagents. A preferred use of the arrays is to implement multiplexbinding assays for the simultaneous detection of two or more analytesusing, for example, flow cytometry or microscopy. For use in suchassays, the microparticles are coated with analyte-specific reagentssuch that microparticles within a population are coated with reagentshaving the same known specificity and microparticles in differentpopulations are coated with reagents having different specificities. Theidentity of the microparticle populations, determined from themicroparticle fluorescence measured in the two detection channels,enables identification of the analyte bound to the microparticle throughthe analyte-specific reagent. One skilled in the art will understandthat detection can be carried out using any of a number of differentassay formats, including sandwich hybridization formats and competitiveassay formats.

Also comprehended by this disclosure are compositions and kits whichinclude a multidimensional array disclosed herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the overlapping emission spectra of multiple dyes, as usedin the arrays described in Examples 2 and 3.

FIG. 2 shows a dot-plot of an array containing 29 distinct populationsof beads, as described in Example 2.

FIG. 3 shows a dot-pot of an array containing 32 distinct populations ofbeads as described in Example 3.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are provided for clarity. Unless otherwiseindicated, all terms are used as is common in the art. All referencecited herein, both supra and infra, are incorporated herein byreference.

As used herein, the terms “microparticles”, “microbeads”, or “beads” areused interchangeably. These terms refer to small particles with adiameter in the nanometer to micrometer range, typically about 0.01 to1,000 μm in diameter, preferably about 0.1 to 100 μm, more preferablyabout 1 to 100 μm, and, for use in flow cytometry, typically about 1 to10 μm. Microparticles can be of any shape, but typically areapproximately spherical (“microspheres”).

Microparticles serve as solid supports or substrates to which othermaterials, such as target-specific reagents, reactants, and labels, canbe coupled. Microparticles can be made of any appropriate material (orcombinations thereof), including, but not limited to polymers such aspolystyrene; polystyrene which contains other co-polymers such asdivinylbenzene; polymethylmethacrylate (PMMA); polyvinyltoluene (PVT);copolymers such as styrene/butadiene, styrene/vinyltoluene; latex; orother materials, such as silica (e.g., SiO₂).

Microparticles suitable for use in the present invention are well knownin the art and commercially available from a number of sources.Unstained microspheres in a variety of sizes and polymer compositionsthat are suitable for the preparation of fluorescent microparticles ofthe invention are available from a variety of sources, including: BangsLaboratories (Carmel, Ind.), Interfacial Dynamics Corporation (Portland,Oreg.), Dynal (Great Neck, N.Y.), Polysciences (Warrington, Pa.),Seradyne (Indianapolis, Ind.), Magsphere (Pasadena, Calif.), DukeScientific Corporation (Palo Alto, Calif.), Spherotech Inc.(Libertyville, Ill.) and Rhone-Poulenc (Paris, France). Chemicalmonomers for preparation of microspheres are available from numeroussources.

As used herein, “microparticle population” refers to a group ofmicroparticles that possess essentially the same optical properties withrespect to the parameters to be measured, such as synthesizedmicroparticles that, within practical manufacturing tolerances, are ofthe same size, shape, composition, and are labeled with the same kindand amount of dye molecules. For example, unlabeled microparticles,microparticles labeled with a first dye at a first concentration,microparticles labeled with the first dye at a second concentration, andmicroparticles beads labeled with a second dye at the thirdconcentration could constitute four distinct bead populations.

The microparticle arrays of the present invention are detected usinginstruments that have the ability to detect fluorescence light emittedin defined frequency ranges, referred to as “detector channels” or“detection channels”. Typically, such instruments contain multiplephotodetectors, such as photomultiplier tubes or photodiodes, and therange of wavelengths detected by each photodetector is determined by theuse of frequency-dependent filters, dichroic mirrors, or otherdispersive elements, as is well known in the art. Alternatively, thesame detector can be used for multiple frequency ranges by changing thedispersive elements during analysis, as is typical in fluorescencemicroscopy.

For identification of microparticles in the arrays of the presentinvention, two detector channels are used that are close enough thatportions of the emission spectrum of each dye falls within each channel.The detector channels can be non-overlapping channels or partiallyoverlapping. The selection of dyes and appropriate correspondingdetection channels is well known and within the ability of one of skillin the art.

Choice of the detection channels will depend on the application and theinstrumentation used. For example, for use in a flow cytometer in whichtwo channels are used to detect microparticle fluorescence and a thirdis used to detect reporter fluorescence, it is advantageous to minimizethe spillover of the microparticle emissions into the reporter channel.Thus, the channels are selected such that the two channels used fordetecting the microparticle emission are spectrally separated from thereporter Is channel, within the constraints imposed by the instrumentand dyes available. Again, the selection of compatible dyes and channelsis well known and within the ability of one of skill in the art.

The term “analyte” is used herein broadly to refer to any substance tobe analyzed, detected, measured, or labeled. Examples of analytesinclude, but are not limited to: proteins, peptides, hormones, haptens,antigens, antibodies, receptors, enzymes, nucleic acids, polysaccarides,chemicals, polymers, pathogens, toxins, organic drugs, inorganic drugs,cells, tissues, microorganisms, viruses, bacteria, fungi, algae,parasites, allergens, pollutants and combinations thereof. It will beunderstood that detection of, for example, a cell, is typically carriedout by detecting a particular component, such as a cell-surfacemolecule, and that both the component and the bacteria as a whole can bedescribed as the analyte.

As used herein an “analyte-specific reagent” or “target-specificreagent” broadly encompasses any reagent that preferentially binds to ananalyte or target of interest, relative to other analytes potentiallypresent in a sample. A target (analyte) and target-specific(analyte-specific) reagent are members of a binding pair, and eithermember of the pair can be used as the target-specific reagent in orderto selectively bind to the other member of the pair. Examples of targetand target-specific reagent pairs include, but are not limited to,antigen and antigen-specific antibody; hormone and hormone receptor;hapten and anti-hapten; biotin and avidin or steptavidin; enzyme andenzyme cofactor; and lectin and specific carbohydrate.

Preferred target-specific reagents are antibodies or fragments thereofthat include an antigen binding site that specifically binds(immunoreacts with) an antigen.

I. Microparticle Array

The array of present invention is comprised of populations ofmicroparticles, wherein each microparticle is labeled with a singlefluorescent dye. The array consists of a plurality of sets ofmicroparticle populations, wherein each set contains a plurality ofmicroparticle populations. Microparticle populations within a set arelabeled, using the same fluorophore, such that each population exhibitsa measurably distinct mean fluorescence intensity. Microparticlepopulations in different sets are labeled with different fluorescentdyes, wherein all of the fluorescent dyes can be excited by the sameexcitation light, the emission spectra of each dye is detectable usingthe same two detection channels, and the relative amount of emissions ineach of the two detection channels is distinguishably distinct betweendifferent dyes.

The microparticles in the array are detected and uniquely identified byexposing the microparticles to excitation light and measuring thefluorescence of each microparticle in each of the two detectionchannels. The excitation light may be from one or more light sources andmay be either narrow or broadband. Examples of excitation light sourcesinclude lasers, light emitting diodes, and arc lamps. Fluorescenceemitted in detection channels used to identify the microparticles may bemeasured following excitation with a single light source, or may bemeasured separately following excitation with distinct light sources. Ifseparate excitation light sources are used to excite the microparticledyes, the dyes preferably are selected such that all the dyes used toconstruct the array are excitable by each of the excitation lightsources used.

For example, a BD FACSCalibur dual laser flow cytometer (BD Bioscience,San Jose, Calif.) has 488 nm and 635 nm excitation lasers that arefocused on the flow stream at spatially discrete regions, and detectionoptics designed to measure light in three detection channels, designatedFL1, FL2, and FL3, following excitation by the 488 nm laser, and afourth detection channel, designated FL4, following excitation by the635 nm laser. In a preferred embodiment described in the examples, FL3and FL4 are selected as the two detection channels used to identify themicroparticle populations. Thus, in this embodiment, one channel, FL3,is measured following excitation by the 488 nm laser and the secondchannel, FL4, is measured following excitation by the 635 nm laser. Theselection of dyes and detection channels in this example was made inview of the configuration of an existing commercial instrument.Alternatively, a flow cytometer could be configured to measure emissionin both FL3 and FL4 following excitation with a single laser.

The resulting fluorescence data from the microparticles preferably isanalyzed by plotting the fluorescence intensity values on atwo-dimensional dot plot, plotting intensity of the two detectionchannels on the two axis, as is routinely used in flow cytometry. Eachpopulation of microparticles will yield a cluster uniquely positioned inthe two-dimensional dot plot. The expected patterns in the dot-plot canbe seen from the following analysis.

Let F_(i1) and F_(i2) be the mean fluorescence measurements of a ithdyed population in fluorescence channels 1 and 2, respectively, andR_(i)=F_(i2)/F_(i1) be the ratio of intensities for the ith population.Because the relative emission in the two channels is a property of thefluorophore's emission spectrum, the ratio of the intensities, for eachdye, is a constant. Thus, in a linear×linear dot-plot in which the meanfluorescence intensities in channels 2 and 1 are plotted on the ordinateand abscissa, respectively, microparticle populations dyed withdifferent amounts of the same fluorophore (i.e., populations within thesame set) will fall on the line F_(i2)=R_(i)·F_(i1), wherein the slopeis equal to R_(i), the ratio of emissions in the two channels.Populations in different sets will fall on lines having different slopes(different ratios of emissions in the two channels) and will display indifferent regions of the dot-plot.

Flourescent intensity data obtained by flow cytometry typically isplotted using log-transformed data. Using the transformed values,log(F_(i2))=log(R_(i))+log(F_(i1)). Thus, on a log×log dot-plot,populations in all sets are expected to fall on lines having the sameslope, but different “y-intercepts”. FIGS. 2 and 3 show data presentedon log x log dot-plots.

II. Fluorophores

Fluorescent dyes (fluorophores) suitable for use in the presentinvention can be selected from any of the many dyes suitable for use inimaging applications (e.g., flow cytometry). A large number of dyes arecommercially available from a variety of sources, such as, for example,Molecular Probes (Eugene, Oreg.) and Exciton (Dayton, Ohio), thatprovide great flexibility in selecting a set of dyes having the desiredspectral properties.

Dyes used in the present invention to label microparticle populations inthe different sets are selected such that the emission spectra of eachdye is detectable using the same two detection channels, and therelative amount of emissions in each of the two detection channels isdistinguishably distinct between different dyes. Selection of candidatedyes can be carried out routinely based on the emission spectra of thedyes. Candidate dyes are then evaluated empirically by dyeingmicroparticle populations using a concentration series of each dye andsubsequently analyzing the results. A suitable subset of the dyedmicroparticles are then selected for use together in a single array.

Depending on the application, the dyes may be selected based onadditional criteria. For example, in embodiments in which themicroparticles are used in a binding assay wherein an additionalreporter fluorophore is used to measure the amount of binding, it isadvantageous to minimize the spillover of the microparticle emissionsinto the channel used to measure the reporter fluorescence. Thedetection channels are selected such that the two channels used fordetecting the microparticle emission are spectrally separated from thereporter channel, and the dyes used are selected to minimize spilloverof the microparticle emissions into the reporter channel.

Examples of fluorophores from which a suitable set can be selectedinclude, but are not limited to,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives such as acridine, acridine orange, acrindine yellow,acridine red, and acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS); N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BrilliantYellow; coumarin and derivatives such as coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine andderivatives such as cyanosine, Cy3, Cy5, Cy5.5, and Cy7;4′,6-diaminidino-2-phenylindole (DAPI); 5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylaminocoumarin; diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives such as eosin and eosin isothiocyanate; erythrosin andderivatives such as erythrosin B and erythrosin isothiocyanate;ethidium; fluorescein and derivatives such as 5-carboxyfluorescein(FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluoresceinisothiocyanate (FITC), fluorescein chlorotriazinyl, naphthofluorescein,and QFITC (XRITC); fluorescamine; IR144; IR1446; Green FluorescentProtein (GFP); Reef Coral Fluorescent Protein (RCFP); Lissamine™;Lissamine rhodamine, Lucifer yellow; Malachite Green isothiocyanate;4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine;pararosaniline; Nile Red; Oregon Green; Phenol Red; B-phycoerythrin;o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrenebutyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron™Brilliant Red 3B-A); rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G),4,7-dichlororhodamine lissamine, rhodamine B sulfonyl chloride,rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine Xisothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloridederivative of sulforhodamine 101 (Texas Red),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine,and tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolicacid and terbium chelate derivatives; xanthene; or combinations thereof.Other fluorophores or combinations thereof known to those skilled in theart may also be used, for example those available from Molecular Probes(Eugene, Oreg.) and Exciton (Dayton, Ohio).

It will be clear to one of skill in the art that the suitability ofparticular dyes or classes of dyes will depend on the method by whichthe microparticles are labeled, as described further, below. Forexample, large fluorescent proteins may be suitable for labelingmicroparticles by binding the dyes to the surface of the microparticle,but likely would not be suitable for internal labeling using bath-dyeingmethods. Suitable candidate dyes can be selected routinely based on thelabeling methods used.

III. Labeling of Microparticles

Fluorescent dyes have been incorporated into uniform microspheres in avariety of ways, for example by copolymerization of the fluorescent dyeinto the microspheres during manufacture (U.S. Pat. No. 4,609,689 toSchwartz et al. (1975), U.S. Pat. No. 4,326,008 to Rembaum (1982), bothincorporated by reference); by entrapment of the fluorescent dye intothe microspheres during the polymerization process; or by non-covalentincorporation of the fluorescent dye into previously preparedmicrospheres (U.S. Pat. Nos. 5,326,692; 5,723,218; 5,573,909; 5,786,219;and 6,514,295; each incorporated by reference). The method of labelingthe microspheres is not a critical aspect of the invention; any methodthat allows the labeling of the microparticles with a controllableamount of dye can be used.

In a preferred embodiment, the fluorescently labeled microspheres of theinvention are prepared by bath dying of microspheres according towell-known methods. Bath dyeing methods are described, for example, inU.S. Pat. Nos. 5,326,692; 5,723,218; 5,573,909; 5,786,219; and6,514,295, which describe bath dyeing methods using a plurality of dyes,which are equally applicable to dyeing microparticles with single dyes.

IV. Methods of Use

The microparticle arrays of the present invention can be usedessentially in any application in which multiplex microparticle arraysare used or are useful, including application in which themicroparticles are used a solid substrates for ligand binding assays oras labeling reagents.

A preferred use of the arrays is to implement multiplex binding assaysfor the simultaneous detection of two or more analytes using, forexample, flow cytometry or microscopy. For use in such assays, themicroparticles are coated with analyte-specific reagents such thatmicroparticles within a population are coated with the reagents of thesame known specificity and microparticles in different populations arecoated with reagents having different specificities. The identity of themicroparticle populations, determined from the microparticlefluorescence, enables identification of the analyte bound to themicroparticle through the analyte-specific reagent.

Analyte detection assays can be carried out using both competitive andnon-competitive formats. Examples of non-competitive assays includesandwich assays in which a second analyte-specific reagent (a reporter)is labeled to facilitate detection of analytes bound to a microparticle.The microparticle array and fluorescently labeled reporter reagents, onefor each species of analyte to be detected, are incubated with a samplecontaining (or suspected of containing) the analytes of interest toallow for the formation of bead-analyte-reporter complexes for eachanalyte present. The resulting complexes are analyzed, preferably byflow cytometry, to identify and, optionally, quantify the analytespresent in the sample. Because the identity of the analyte bound to thecomplex is indicated by the identity of the bead, multiple analytes canbe simultaneously detected using the same fluorophore for all reporterreagents.

In a competitive assay, the sample suspected of containing analyte isincubated with the microparticle array and an analyte-analogue that iscapable of competing with the analyte for the limited number of bindingsites provided by coated microparticle. In one embodiment,analyte-analogue labeled with a reporter fluorophore is provided in aconcentration sufficient to saturate the binding sites on themicroparticle. The presence of analyte, which competes with and therebyreduces the number of labeled analyte-analogues bound to themicroparticle, results in a measurable decrease in the reporterfluorescence associated with the microparticle.

In an alternative embodiment, microparticle arrays of the presentinvention are useful as labeling reagents. The microparticles in eachpopulation are coated with an target-specific reagent, wherein thetarget is any molecule to be labeled. The sample is incubated with thearray for a time sufficient to allow the target molecules to bind to thetarget-specific reagent coating the microparticles, thus labeling themolecules. The use of the multidimensional arrays of the inventionenables labeling a large number of distinct target molecules whilerequiring only two detection channels to uniquely identify the labels.In a preferred embodiment, microparticle arrays of the present inventionare used as labeling reagents in fluorescence microscopy.

Methods for attaching an antibody or other target-specific reagent to amicroparticle are known in the art. Commercially availablemicroparticles typically are provided with amino groups or carboxylgroups to facilitate the covalent attachment of antibodies using wellknown chemistry. However, any method used by those skilled in the artmay be employed.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed.

Example 1 Preparation a Microparticle Array

This example describes preferred methods for preparing a microparticlearray for use in flow cytometry.

The array described herein is intended for use in a detection assay inwhich the microparticles are coated with an analyte-specific reagent,and a labeled-reporter reagents are used to measure the amount ofanalyte bound to a microparticle. To maximize sensitivity of thedetection assay, it is desirable to label the reporter reagents with aparticularly bright fluorophore, preferably phycoerythrin (PE), whichhas an emission maximum at 578 nm. Thus, the array is prepared assumingthat an appropriate detection channel is reserved for measuring thePE-labeled reporter reagents.

1. Detection Channels

The detection channels used to measure the microparticle fluorescenceare selected from the channels available using a flow cytometer.Preferred flow cytometers are the BD FACSCalibur™ flow cytometer and theBD FACSArray™ flow cytometer (BD Biosciences, San Jose, Calif.). Thefilters, which define the detector channels, differ slightly between thetwo instruments (in the standard, commercially availableconfigurations).

A BD FACSCalibur dual laser flow cytometer has 488 nm and 635 nmexcitation lasers. Fluorescence following excitation by the 488 nm laseris measured in the following detection channels:

FL1: 530/30 nm (515-545 nm)

FL2: 585/42 nm (564-606 nm)

FL3: 670 nm Long Pass (>670 nm)

Fluorescence following excitation by the 635 nm laser is measured in thefollowing detection channel:

FL4: 661/16 nm (653-669 nm)

Using a BD FACSCalibur flow cytometer, the FL2 channel is used formeasuring the PE-labeled reporter, and microparticle populations areidentified from the emissions in the FL3 and FL4 detector channels.

The BD FACSArray flow cytometer has 532 nm and 635 nm excitation lasers.Fluorescence following excitation by the 532 nm laser is measured in thefollowing detection channels:

Yellow: 585/42 nm (564-606 nm)

Far Red: 685 nm long pass (>685 nm)

Fluorescence following excitation by the 635 nm laser is measured in thefollowing detection channels:

NIR (Near Infra Red):780/60 nm (750-810 nm)

Red: 661/16 nm (653-669 nm)

Using a BD FACSArray flow cytometer, the Yellow detection channel isused to measure the PE-labeled reporter reagent, and bead populationsare identified from the emissions in the Red and Far Red detectorchannels.

2. Dyes

Candidate dyes are selected in a routine manner based on their emissionspectra such that the all the dyes emit in the two detection channelsselected for use in identifying bead populations, as described above.Additionally, the dyes are selected such that the emission of the dyesin the PE-detection channel (FL2 or Yellow) is minimal. Preferred setsof preferred dyes are described in the following examples.

3. Preparation of Microparticles

Suitable undyed microparticles for use in constructing an array are 7.7micron, 10% solid polymeric beads from Bangs Laboratories (Carmel,Ind.). Prior to dyeing, the beads are prepared as follows. The beads arewashed 3 times in 15 ml MeOH. The beads are then washed in 15 ml ofanhydrous MeOH (MgSO₄-treated) and then suspended in 17.5 ml ofanhydrous MeOH. For dyeing, 1 ml portions (3.86×10⁷ beads) are put intoseparate tubes, each containing a small stirring bar, and stirred.

4. Preparation of Dyeing Solutions

For each dye, a series of 1.0 ml dyeing solutions are preparedconsisting of the dyeing reagents in the proportions shown in the table,below.

Dye Tube 30% MeOH/70% EtOH 1,4 Dioxane (0.1 μmole/μl in DMF) A 500 μl420 μl 80 μl B 540 μl 420 μl 40 μl C 560 μl 420 μl 20 μl D 570 μl 420 μl10 μl E 575 μl 420 μl  5 μl F 577.5 μl   420 μl 2.5 μl  G 578.75 μl  420 μl 1.25 μl   H 579.4 μl   420 μl 0.6 μl Alternatively, another suitable organic solvent, such as CH₂Cl₂, can beused in place of 1,4 Dioxane.5. Bead Dyeing

Each of the 1 ml dyeing solutions is added to a separate tube containinga 1 ml preparation of washed beads while stirring vigorously at roomtemperature. The bead/dye mixtures are stirred at 50° C. for 1 hour inthe dark to allow the dye to be absorbed into the beads.

After dyeing, 10 ml of MeOH are added to each tube. The tubes arevortexed and then centrifuged at ˜3,000 rpm for 4 minutes to pellet thedyed beads. The pellets are washed 2 times with 5 ml MeOH, then washedwith 10 ml of 0.05% Tween-20, 0.1% NaN₃, then suspended in 3 ml of 0.05%Tween-20, 0.1% NaN₃. The concentration of the resulting dyed beadsolution is approximately 10⁴/μl(10⁷/ml).

6. Testing and Selection of Bead Populations

Dyeing is carried out as described above using each of the candidatedyes to generate a plurality of bead sets, each set dyed with adifferent candidate dye, and each set containing a series of beadpopulations dyed with different concentrations of the same dye. Theplurality of bead sets represents a candidate array from which asuitable array is selected. The selection of a suitable array isdetermined empirically, and will depend on a number of parameters, suchas the dyes selected and the dyeing efficiencies.

To select an suitable array from the candidate array, bead populationsare analyzed on either a BD FACSCalibur™ flow cytometer or a BDFACSArray™ flow cytometer using the detector channels described above,and the fluorescence intensity data from each bead in each of the twodetector channels are plotted in a two-dimensional dot-plot. Beadpopulations that form well-defined clusters that are simultaneouslyon-scale (i.e., the data not compressed against an edge of the dot-plot)and essentially not overlapping with other bead populations are selectedfor use in the combined array.

Additionally, the emission from the beads is measured in the PE-channel(FL2 or Yellow) to determine the amount of spillover into the channelused to measure the PE-labeled reporter molecules. Preferably, thespillover into the PE-channel is minimal so as to minimize interferencein the measurement of the reporter reagents.

Because the selection of bead populations to use in the array isempirical, bead dyeing and analysis is expected to be an iterativeprocess in which beads are incrementally added to an existing array.Appropriate adjustments can be made to the dyeing procedure in a routinemanner based on the experimental results. For example, if mostpopulations within an initially dyed set are off scale because the dyeintensity is too high, such that only a subset was selected for use inthe final array, additional populations of beads can be dyed using alower concentration of dye. Subsequent analysis with the first dyedpopulations selected for inclusion in the array will allow for extendingthe size of the array. Furthermore, an additional set of beadpopulations, dyed with a distinct dye, can be added incrementally to anexisting set to extend the size of the array.

Example 2 29-Population Array

Bead subsets were prepared essentially as described in Example 1 usingeach of the dyes listed in the table, below. All dyes were obtained fromExciton (Dayton, Ohio). Excitation and emission maxima, which weremeasured in either ethanol, methanol, or dichloromethane, are known tobe somewhat dependent on the solvent used for the measurements, andslightly different results may be obtained using different solvents.

Dye Excitation Max Emission Max Populations used in array LD700 647 6737 LDS730 614 695 4 LDS750 572 704 3 LDS751 542 700 1 Oxazine 725 645 6766 ABS643 640 655 8

The emission spectra of these dyes (with the exception of LDS751) areshown in FIG. 1. Also shown are the boundaries of the detection channeldefined using a BD FACSArray flow cytometer.

Sets of dyed beads, each containing bead populations dyed with differentamounts of one of the dyes, were combined and were analyzed on a BDFACSArray flow cytometer, as described above. Bead populations that wereboth uniquely distinguishable and simultaneously on-scale in a FarRed×Red dot-plot were selected empirically for use in the finalmultiplex bead set. FIG. 2 shows a dot-plot (Far Red×Red) of theselected bead array containing 29 distinguishable bead populations. Thenumber of bead populations used in the final set from each of thesubsets is indicated in the table, above.

The emissions of the bead populations in the PE-channel (Yellow) alsowere measured (results not shown), and minimal spillover was observedfor all populations.

Example 3 Extended Array

The bead array described in Example 2 was incrementally extended by theinclusion of a bead set dyed using LDS765 (Exciton, Dayton, Ohio),prepared essentially as described above. The measured excitation andemission maxima for the dye are shown in the table, below:

Dye Excitation Max Emission Max Populations used in array LD765 595 7523

For this analysis, the sets of dyed beads, were analyzed on a BDFACSCalibur flow cytometer, as described above. Bead populations fromthe set dyed with LDS765 were selected such that the populations wereboth uniquely distinguishable and simultaneously on-scale relative tothe bead populations in the previously constructed array. Threepopulations could be added to the 29-population array. FIG. 3 shows adot-plot (FL3×FL4) of the selected bead array containing 32distinguishable bead populations.

The emissions of the bead populations in the PE-channel (FL2) also weremeasured (results not shown), and minimal spillover was observed for allpopulations.

1. A method of measuring a plurality of analytes in a sample comprising:a) contacting a sample with an array of populations of microparticles,comprising i) a plurality of sets, wherein each set contains a pluralityof populations of microparticles, wherein microparticle populationswithin a set are labeled with different levels of a single fluorophoresuch that each population exhibits a measurably distinct fluorescenceintensity; wherein microparticles in different sets are labeled withdifferent fluorophores; wherein all of said fluorophores haveoverlapping emission spectra and emit light that is detectable using asingle pair of detection channels, and the relative emission in each ofthe two detection channels is distinguishably distinct between differentfluorophores; wherein each of said microparticle populations isdistinguishable by its fluorescence emissions in said pair of detectionchannels; and wherein said array comprises at least 3 sets ofmicroparticle populations; and ii) a plurality of analyte-specificreagents, wherein reagents of the same specificity are coupled tomicroparticles in the same population, and reagents of differentspecificity are coupled to microparticles in different populations; fora time sufficient to allow for the formation of microparticle-analytecomplexes; b) exposing said microparticle-analyte complexes toexcitation light; c) measuring fluorescence of each of saidmicroparticle-analyte complexes in each of said pair of detectorchannels; d) identifying the specificity of said analyte-specificreagents coupled to said microparticles, thereby identifying the analytepresent in each of said microparticle-analyte complexes; e) measuringthe amount of analyte present in each of said microparticle-analytecomplexes, to provide a measure of the plurality of analytes present insaid sample.
 2. The method of claim 1, wherein said sample is furthercontacted with a set of fluorescently labeled analyte-specific reporterreagents, wherein said set contains an analyte-specific reporter reagentspecific for each analyte to be measured.
 3. The method of claim 1,wherein said detecting is carried out by flow cytometry.
 4. The methodof claim 1, wherein at least one of said analyte-specific reagents is anantibody that binds specifically to an analyte that is an antigen, or atleast one of said analyte-specific reactants is an antigen that bindsspecifically to an analyte that is an antibody.
 5. The method of claim1, wherein said array comprises at least 4 sets of microparticlepopulations.
 6. The method of claim 1, wherein said array comprises atleast 5 sets of microparticle populations.
 7. The method of claim 1,wherein said array comprises at least 20 populations of microparticles.8. The method of claim 1, wherein said array comprises at least 30populations of microparticles.